Plant cell culture is currently being studied as an alternative to using transgenic plants, microorganisms, yeast cells, or insect and mammalian cell cultures for recombinant protein production (Magnuson et al., Protein Expres. Purif. 13, 45–52 (1998); Fischer et al., Biotechnol. Appl. Biochem. 30, 109–112 (1999); Doran, P. M., Curr. Opin. Biotech. 11, 199–204 (2000); James et al., Protein Expres. Purif. 19, 131–138 (2000)). Some advantages of using plant cell suspension cultures for production of biologically active compounds are low raw material costs, capability of post-translational modifications, and diminished risk of mammalian pathogen contamination.
One of the major drawbacks of plant cell culture production systems is the slow growth rate of plant cells. Traditional batch cultures include a long lag time to allow growth of plant cells to productive levels. After maximal cell growth occurs, the cells are harvested with the product and the process begins again.
Studies using the constitutive CaMV 35S promoter in transgenic tobacco have observed improved results with semi-continuous and continuous systems versus batch systems for production of extracellular foreign proteins (Des Molles et al., J. Biosci. Bioeng. 87, 302–306 (1999); Ryland et al., J. Microbial. Biotech. 10, 449–454 (2000)). Intermittent medium exchanges have also been implemented using wildtype plant cultures where secondary metabolite production was elicited (Scragg et al., Enzyme Microb. Tech. 12, 292–298 (1990); Su et al., Biotechnol. Bioeng. 42, 884–890 (1993); Su et al., Appl. Microbiol. Biot. 44, 293–299 (1995)). Since these secondary metabolites were not secreted, they could only be harvested once a maximum concentration was reached intracellularly. Therefore, the effects of revitalizing and reusing the plant cells for subsequent growth and expression phases could not be evaluated. However, recent research has demonstrated higher yields of secreted taxol in wildtype plant cultures using re-elicitation and periodic medium renewal compared to single batch type cultures (Phisalaphong et al., Biotechnol. Prog. 15, 1072–107 (1999); Wang et al., Appl. Microbiol. Biot. 55, 404–410 (2001)).
Inducible promoters allow regulated gene expression, and therefore independent control and optimization of the growth and product expression phases. Traditional batch culture is not well suited to the use of inducible promoters because of the lag time and expense of restarting the culture with each harvest. A continuous culture is also undesirable for an inducibly regulated promoter since it would be difficult to implement rapid changes in the concentration of regulatory molecules.
There has been little research involving maximizing productivity in plant cell cultures by applying the benefits of an inducible promoter to the long term, semi-continuous production of a secreted recombinant protein. This type of operation can help make plant cell culture economically attractive for recombinant protein production compared to other hosts or transgenic plants, depending on production level and downstream processing costs.
Previous work demonstrated production of a human therapeutic protein, recombinant α1-antitrypsin (rAAT), in transgenic rice (Oryza sativa L.) suspension cultures (U.S. Pat. No. 6,127,145). Use of an inducible rice α-amylase (RAmy3D) promoter to direct expression of a heterologous protein and use of a rice α-amylase signal peptide to direct secretion of a heterologous protein into the culture medium have been disclosed. (Huang N. et al., Plant Mole. Biol. 23, 737–747 (1993); Rodriguez, WO 95/14099.) The RAmy3D promoter and signal peptide were originally disclosed in Huang N., et al., Nucl. Acids Res. 18, 7007–7014 (1990). Production of rAAT in transgenic rice cultures has also been demonstrated by Terashima and coworkers (Terashima et al., Appl. Microbiol. Biot. 52, 516–523 (1999); Terashima et al., Biochem. Eng. J. 6, 201–205 (2000); Terashima et al., Biotechnol. Prog. 17, 403–406 (2001)).
Maximization of protein productivity from plant tissue culture cells would be assisted by semi-continuous operation combined with identification of optimal conditions for cell growth and protein expression. Semi-continuous large scale growth of plant cells occurs over a long time period, thereby increasing the risk of contamination of the culture. Currently, optimal conditions for growth and expression of protein are identified by techniques that require sampling of the culture and time-consuming off-line analysis, such as viable cells counts and immunological identification of protein products. Sampling of the culture is undesirable due to the potential to introduce microbial contaminants from the surrounding environment as the sample is taken. Contamination would render the batch unusable. In addition, the optimal time for performing medium exchanges could be missed due to the length of time required for off-line analysis. Identification of easily assayable variables or on line measurements that correlate with vigorous cell growth or high levels of protein expression would increase the effectiveness of this method of protein production. The present application addresses these and other needs.